The present invention generally relates to methods for measuring coagulation of blood and blood products, and more particularly, to the use of an inhibitor to minimize blood product coagulation in vitro, and obviate sources of inconsistency and errors in blood coagulation. The present invention has a variety of uses, e.g., prolonging plasma clotting times, optimizing sensitivity of plasma coagulation assays, and enhancing storage of blood and blood products such as plasma.
Blood coagulation (clotting) assists homeostasis by minimizing blood loss. In vivo, clotting usually requires vessel damage, platelet aggregation, coagulation factors and inhibition of fibrinolysis. The coagulation factors have been reported to act through a cascade that relates vessel damage to formation of a blood clot. See generally L. Stryer, Biochemistry, 3rd Ed, W. H. Freeman Co., New York; A. G. Gilman et al., The Pharmacological Basis of Therapeutics, 9th Edition, McGraw Hill Inc., New York, pp.1341-1359; and Mann, K. G. et al. (1992) Semin. Hematol. 29:213.
Initiation of blood coagulation arises from two distinct pathways: the intrinsic (contact) and extrinsic pathways. The intrinsic pathway can be triggered in vitro by contact of blood borne factor with artificial negatively charged surfaces such as glass. In contrast, the extrinsic pathway can be initiated in vivo or in vitro when tissue factor (TF) on a phospholipid surface, normally sequestered from the circulatory system, comes into contact with blood following injury. Both pathways are characterized by the assembly of multiple protein complexes on procoagulant surfaces, which serves to localize the response to the site of injury. See e.g, Mann, K. G. et al. (1990) Blood 76: 1; Mann, K. G. et al. (1992), supra.
Current theories of coagulation maintain that interplay between the two pathways is required for efficient blood clotting. See S. I., Rapaport and L.V.M. Rao (1995) Throm. Haemost. 74: 7; and Stryer, L. supra and references cited therein.
The contact pathway has been further divided into early and late steps. These steps are typically associated with specific coagulation factors. For example, the early contact pathway is associated with activated prekallikrein and Factor XII, whereas the late contact pathway involves Factors VIII and IX. It has been reported that hemophilia A, B and C are each correlated with deficiencies in the late contact pathway (Factor VIII, Factor IX, and Factor XI, respectively). Hemorrhagic tendencies have not been found for prekallikrein or factor XII deficiency. Accordingly, these early contact factors are not thought to be relevant for initiation and maintenance of coagulation. See e.g., Davie, E. W. et al. (1991) Biochem. 30:10363.
Many activities of the extrinsic and intrinsic tenases (factor VIIIa-factor IXa) and the prothrombinase complex are facilitated by activated platelets and other phospholipid membranes. Characterization of the impact of therapeutic agents in vivo is usually analyzed by methods in which the contact pathway is attenuated or eliminated. See Nemerson, Y. (1988) Blood 71:1, Rand, M. D. et al. (1996) Blood 88: 1; and Monroe, D. M. et al. (1994) Brit. J of Haemot. 88: 364.
There have been attempts to understand how blood coagulation is initiated and controlled. One approach has been to reproduce blood coagulation as it is thought to occur in vivo. For example, by analyzing reactions associated with blood coagulation in vitro, it has been possible to detect relationships between certain blood coagulation factors. Specific attempts have involved analysis of fractionated blood and particularly blood products such as plasma. Current in vitro models of blood clotting focus on the activity of specific blood factors. See e.g., Davie, E. W. et al. supra.
Confusion about the role of these pathways in coagulation has arisen from several difficulties such as in processing, storing, and studying blood and blood products. Untreated whole blood or blood products such as plasma typically coagulate within minutes. The clotting can be reduced or eliminated by addition of a calcium-chelating agent such as citrate. In particular, citrate has been reported to interfere with the assembly and function of prothrombinase and extrinsic and intrinsic tenases. Citrated blood can be stored in liquid form for a limited period of time (e.g., days to weeks) , or can be manipulated to produce blood products such as blood cell isolates, platelet rich and platelet poor plasma. Plasmas that include citrate can be stored for extended periods (months to years) by freezing at temperatures below about xe2x88x9270xc2x0 C. In most instances, the plasma is recalcified for use. However, recalcified plasma will typically clot spontaneously due to contact activation in most storage vessels, where contact activation can occur within about 2 to 4 minutes. As a result, most coagulation assays are usually performed on citrated plasma fractions that have been frozen for storage then thawed. However, such fractions cannot be recalcified until immediately prior to use.
Coagulation tests are often performed either on blood or blood products. In simple tests such as bleeding time tests, wounds are made in a patient and the time until clot formation is noted. Additionally, whole blood coagulation tests have been devised by drawing blood directly into a tube, then rocking or agitating until a clot is observed. Such tests are not very informative, as the sources of initiation are not well controlled and comparisons among patients are difficult. In clinical settings, citrated plasma isolates are the most widely used blood product for coagulation testing, due to prominence of the prothrombin time (PT) and activated partial thromboplastin time (aPTT) tests. The PT is the more convenient assay, and is performed by addition of a large quantity of thromboplastin to the citrated plasma, with subsequent initiation of the reaction by calcium addition. The time to clot formation is noted, which for most normal donors is typically about 10 to about 14 seconds. The aPTT test involves about a 3 to about 5 minute preincubation of the citrated plasma with a mixture of phospholipids and solids possessing negatively charged surfaces. The reaction is initiated by calcium addition, and the clot time for normal donors typically falls between 25 and 43 seconds. While well established in the clinical venue, neither assay is entirely suitable to mimic the physiological coagulation reaction in its entirety. See generally Williams Hematology, infra.
For example, while the PT measurement employs the physiologically relevant initiator TF and the assay is sensitive to Factors V, VII, X, and prothrombin (II), the concentration used is sufficiently high that the reaction is usually insensitive to deficiencies or abnormalities in coagulation Factors VIII or IX. Clotting occurs rapidly in normal individuals (about 10 to about 14 seconds), and errors in measurement on the order of seconds are a significant fraction of the total clot time. When the assay is used to monitor administration of anti-coagulants, the target range for prolongation of the clot time is between about 2.5 to about 3.5 times normal, or between about 25 and 49 seconds. There has been recognition that this time range is often too small to permit accurate analysis.
The aPTT assay is also associated with problems. For example, since initiation proceeds through the early contact pathway members, Factor VII is bypassed in this reaction. As a result, this assay in insensitive to deficiencies or abnormalities in this biologically important coagulation factor. For this reason, the aPTT is not typically considered suitable for monitoring anti-coagulation by coumadin or other warfarin derivatives which strongly affect the ability of Factor VIIa to serve as an initiator of the coagulation reaction.
Additionally, most aPTT assays use plasma and are not compatible with whole blood. Thus, a source of phospholipid must often be provided, and the contributions of platelets and other cells or inhibitors of cellular processes cannot be assessed. Certain whole blood clotting assays and particularly the activated clotting time (ACT) are reported to be responsive to antiplatelet regimens. See e.g,. Moliterno, D. J. et al. (1995) Am. J. Cardiol. 75: 559; Ammar, T. et al. (1997) Circulation 95:614.
Furthermore, recalcified plasma will typically clot spontaneously owing to the contact pathway. Supraphysiologic concentrations of tissue factor must often be added to perform the PT with reproducible results. The resulting rapid time to clot (less than about 15 seconds) eliminates the contribution of the intrinsic tenase and substantially limits assay sensitivity to a variety of therapeutic agents. See e.g., Schultz, N.J. (1991) Pharmacotherapy 11: 312.
Although there has been some progress toward controlling coagulation of blood, plasma and other blood products in vitro, there is a need for improved methods for storage of these materials. For example, while citrate and other calcium chelators such as ethylene diamine tetraacetate (EDTA) are usually effective attenuators of prothrombinase and tenase assembly and function, these chelators have little effect on the early contact reactions involving Factor XII, Factor XI and prekallikrein activation which often proceed unchecked during storage. Other methods have been developed involving use of certain charged polymers such as heparinoids. These compounds are usually effective in reducing spurious clotting in blood and blood products. However, these compounds have drawbacks as well. See e.g., A. G. Gilnan et al. supra.
These and related problems have also hampered whole blood coagulation assays. For example, prior practice has dictated rapid analysis of the whole blood within minutes of sampling. One approach has been to prepare plasma from the whole blood to facilitate analysis at a later time. However as noted, it has been difficult to perform assays which are sensitive, reproducible, and produce accurate models of in vivo coagulation. See also Osterud, B. et al. (1977) PNAS (USA) 74:5260.

There has been some progress toward controlling whole blood coagulation in vitro. For example, corn trypsin inhibitor (CTI) has been used to reduce whole blood coagulation in vitro. See e.g., Rand, M.D. (1996) et al. Blood, 88: 3432; Hojima,Y. et al. (1980) Thromb. Res., 20: 149; and Munakata, M. et al. (1996) Rinsho Byori (Japan), 44: 883.
However, prior use of CTI has been associated with drawbacks. For example, it has been unclear whether CTI can inhibit clotting of plasma and other blood products. In particular, there has been recognition that fractionation or prolonged storage of whole blood may adversely impact the capacity of CTI to prolong clotting times. Additionally, some blood products and especially plasma are routinely subjected to multiple cycles of freezing and thawing. Those cycles may also impact CTI effectiveness. Moreover, CTI may not always inhibit clotting of whole blood or blood products in a reproducible manner. These and other considerations have limited use of CTI as a coagulation inhibitor.
It would be desirable to have efficient and reproducible methods for measuring coagulation of whole blood and blood products such as plasma that more closely resemble in vivo coagulation. It would be especially desirable to have tissue factor initiated plasma coagulation assays that exhibit prolonged clotting times and enhanced sensitivity to late contact pathway factors and particularly Factors VIII and IX.
The present invention features methods for measuring coagulation of blood and blood products, and more particularly, to the use of a specific inhibitor to minimize blood product coagulation in vitro, and to obviate sources of inconsistency and errors in blood coagulation. In one aspect, the methods include adding corn trypsin inhibitor (CTI) to plasma in an amount sufficient to enhance coagulation analysis. The methods have several important uses, e.g., to increase the sensitivity and reproducibility of plasma coagulation assays, and to prolong lifetimes of blood cell or plasma isolates.
Additionally provided by this invention are methods for inhibiting coagulation of blood or a blood product. Preferred blood is whole or minimally altered blood from a mammal and particularly a human patient. A preferred blood product is plasma such as frozen plasma or other plasmas as described herein. In one embodiment, the methods significantly reduce or eliminate coagulation in the blood or blood product sufficient to provide for enhanced coagulation analysis or storage of the blood or blood product. Especially preferred methods-employ an effective amount of corn trypsin inhibitor (CTI) to inhibit coagulation of the blood or blood product The CTI can be used by itself as a sole anticoagulant or the CTI can be used in combination with a sufficient amount of at least one other anticoagulant as described below..
In particular, we have discovered that CTI can be used to provide enhanced and more accurate results in clotting analysis both in whole blood and blood products such as plasma. In one aspect, the invention features methods for substantially prolonging clotting of a blood product by adding a sufficient amount of CTI to reduce or eliminate intrinsic (contact) coagulation. In one embodiment, the CTI inhibits plasma clotting time by facilitating a significant reduction in contact coagulation. Preferably, the amount of added CTI is sufficient to inhibit the plasma clotting time in recalcified plasma in excess of between about 1000 to about 3600 seconds or longer when compared with a suitable control in the same coagulation assay. Additionally preferred use of the methods provide plasma clotting times wherein the desired International Normalized Ratio (INR) is between about 1 and about 3.5 and in some instances up to about 6.5. The substantially prolonged clotting times achievable by the present methods substantially enhances the sensitivity and reproducibility of a variety of coagulations assays such as those described below.
By reference herein to a xe2x80x9cblood productxe2x80x9d or like term is meant a purified composition derived from whole blood from a mammal and especially a human patient. Preferably, at least one blood component (e.g., blood cells, blood factors, or blood related proteins) has been removed from the whole blood. Particular blood products are deficient in at least one specific cell type such as red blood cells, white blood cells (immune cells), and platelets. A preferred blood product is plasma, ie. the fluid portion of blood. A particular type of plasma is referred to as xe2x80x9cplatelet poorxe2x80x9d or xe2x80x9cplatelet deficientxe2x80x9d plasma.
The amounts or specific activity of CTI used in the present methods varies depending on several parameters such as intended use and more specifically to the degree of inhibition desired. However, in most instances the amount or specific activity of the CTI will be sufficient to prolong the clotting time of a desired blood product by between about 100 to about 2000 seconds or longer up to about 3600 seconds when compared to a control in the same coagulation assay. In a specific embodiment, the amount of CTI used will be sufficient to prolong plasma clotting time and particularly the clotting time in platelet-deficient plasma as determined by a suitable coagulation assay.
We have also found that CTI extends the useful range of certain modified coagulation assays such as those described below. This extension is desirable to ensure responsiveness to blood coagulation factors and particularly Factors VIII and IX in certain assays. In particular, plasma clotting assays that mimic the physiological coagulation reaction will be improved by sensitivity to levels of the Factors VIII or IX in the range of between about 0 units/ml to about 1 unit/ml up to about 2 units/ml or more.
Importantly, it has also been found that the CTI provides for more uniform and reproducible analysis of coagulation especially using plasma. The sensitivity and reproducibility of the present methods makes them especially useful for characterizing blood disorders associated with abnormal levels of these and other coagulation factors including prothrombin Factor V, Factor VII, Factor X and Factor XI.
Without wishing to be bound by any theory, it is believed that CTI improves clotting assays by reducing or eliminating adverse effect of the contact pathway, the effect of which can vary depending on the compositions of the samples and the nature of the surfaces those samples contact. Importantly, it is also believed that use of CTI in accord with the present invention makes certain coagulation assays more representative of in vivo coagulation reactions.
Specific use of the methods involves adding plasma and particularly platelet-deficient plasma to a reaction vessel comprising a suitable amount of CTI. Preferred are methods in which the reaction vessel further includes at least one suitable anti-coagulant such as a buffered calcium-chelating compound. The CTI and anti-coagulant can be provided in any suitable form such as a pre-determined amount of a liquid, suspension or lyophilized material. More specific examples of anti-coagulants suitable for use with the present invention are provided below.
We have also found that CTI can be used to facilitate storage of whole blood or a blood product at reduced temperature e.g., about freezing or below freezing temperatures. More specifically, by supplementing the whole blood or blood product with an amount of CTI sufficient to reduce or eliminate contact coagulation, effective and reproducible analysis of the whole blood or blood product is more readily obtained. In particular, the CTI has been found to remain a potent anti-coagulant even if the blood or blood product is subjected to one or more freeze-thaw cycles.
Accordingly, in one aspect, the invention features methods for storing blood or a blood product which methods involve contacting same with a suitable amount of CTI. In one embodiment, the methods involve freezing blood or the blood product in the presence of the CTI, e.g., by adding the CTI to a freezing or previously frozen blood or blood product. In a specific embodiment, the CTI can be layered on top of the frozen blood or blood product. In this instance, the frozen blood or blood product exhibits substantially prolonged clotting time when subjected to a clotting permissive temperature. Significantly, the methods can be used to improve storage of whole blood or blood products such as plasma by decreasing or eliminating intrinsic coagulation if the whole blood or blood product is subjected to a clotting permissive temperature.
Further provided are methods for measuring clotting in blood or blood products and especially plasma. In one aspect, the methods involve adding a suitable amount of CTI to suitable coagulation assay to facilitate prolonged clotting. For example, the invention can be used with a PT assay with samples obtained from a patient receiving anti-coagulant therapy. In a more specific embodiment, the CTI can be used in a PT assay with platelet poor plasma. The CTI can be used to reduce or eliminate contact coagulation-in other assays such as the dilute thromboplastin assay or those specific assays sensitive to Factors VIII, IX or XI described below.
In one embodiment, the methods include treating a blood product with at least one anti-coagulant, e.g., a calcium-chelating agent or heparinoid, and then subjecting the blood to conditions conducive to making the blood product. In a more specific embodiment, the blood product is plasma and the conditions include conventional filtration or centrifugation steps to reduce or eliminate unwanted blood cells from the blood. In this embodiment, the plasma is also contacted with an amount of CTI sufficient to inhibit or eliminate contact coagulation. In cases in which the anti-coagulant is a calcium-chelating agent, the plasma is preferably recalcified. The recalcified plasma can then used in a desired coagulation assay.
If desired, the method can be modified by adding a suitable amount of CTI to the blood before or after treating with the anti-coagulant(s).
Also provided by this invention are novel blood clotting assays that are suitably adapted for use with whole or minimally altered blood. The assays have a wide spectrum of important uses including what is sometimes referred to as xe2x80x9cpoint-of-carexe2x80x9d analysis. That is, preferred blood clotting assays are compatible with bedside use, e.g., in a hospital, outpatient or home setting. This feature of the invention facilitates analysis of blood coagulation essentially in xe2x80x9creal-timexe2x80x9d, thereby improving patient management and enhancing assessment of therapeutic agents in the blood. More particular blood clotting assays are sensitive to relevant plasma and cellular events involved in blood coagulation including management by pharmacological agents. In preferred blood clotting assays, coagulation is initiated with low concentrations of lipidated tissue factor while the contact pathway is suppressed with CTI. Significantly, the assay detects the activity of a wide variety of agents such as anticoagulants and especially antithrombotic and/or antiplatelet agents with increased sensitivity. The assay also facilitates detection of additive and synergistic effects of combinations of the agents. Additional uses and advantages of the point-of-care blood clotting assay are discussed below and in the Examples.
Additionally provided are methods for assaying clotting of blood products that have been subjected to freezing or below freezing conditions. As noted, use of CTI in accord with the present invention can provide potent anti-coagulant activity that is significantly resistant to one or multiple freeze-thaw cycles. In one aspect, the methods include treating whole blood with at least one anti-coagulant, preferably a calcium-chelating agent or heparinoid, and then subjecting the whole blood to conditions conducive to making the blood product. In one embodiment, the blood product is plasma and the treated whole blood is subjected to particular conditions described herein for making plasma. In this embodiment, the plasma is frozen and then contacted with a suitable amount of CTI. In a particular embodiment, a suitable amount of CTI is added to the frozen plasma in an amount sufficient to inhibit or eliminate contact coagulation. In another embodiment, the CTI is mixed with the plasma prior to or during freezing. In either chase, contact coagulation of the plasma can be significantly reduced or eliminated if the plasma is subjected to a clotting permissive temperature. In instances where the anti-coagulant is a calcium-chelating compound such as a citrate salt, thawed plasma is recalcified. The recalcified plasma can then be used in any suitable coagulation assay.
Also provided is what is sometimes referred to herein as an Extended Plasma Prothrombin Time (XpPT) assay. In one embodiment, the assay can be used to suitably measure coagulation in blood products such as plasma and especially frozen or previously frozen plasma. As discussed below, the assay is very flexible and has a number of important applications including use as a convenient tool for implementing nearly universal hemostasis management. More particularly, the assay can be used to monitor and quantify (if desired) clotting reactions indicative of abnormal bleeding such as those associated with congenital bleeding disorders. The assay is also useful for monitoring the impact of anti-coagulant therapy. Significantly, the assay can be titrated in accord with the I.S.I., thus leading to a nearly universally equivalent anticoagulant monitoring system that is usually independent of thromboplastin source. This advantage of the invention reduces or eliminates need to xe2x80x9cstockpilexe2x80x9d blood coagulation reagents, thereby making analyses more cost effective and easier to perform. As discussed below, the assay can be used to optimize xe2x80x9cdifficult-to-standardizexe2x80x9d reagents such as thromboplastin.
The above-described methods can be modified according to intended use. For example, a suitable amount of CTI can be added to the whole blood prior to making the desired blood product. Preferably, the CTI is added in an amount sufficient to inhibit or eliminate coagulation of the blood or the blood product.
The invention features additional methods for significantly reiducing or eliminating contact clotting in a suitable coagulation assay. In one aspect, the methods include treating whole blood with a suitable amount of CTI and at least one other anti-coagulant, preferably a calcium-chelating agent or heparinoid. The treated whole blood is then subjected to conditions conducive to making the blood product. In a one embodiment, the blood product is plasma. In instances where the anti-coagulant is a calcium-chelating compound such as a citrate salt, the plasma is recalcified. The recalcified plasma can then be used in a suitable coagulation assay.
The present invention features significant advantages. For example, specific methods described herein involve a suitable coagulation assay to which CTI is added. In this example, the assay can utilize tissue factor (TF) and especially relipidated TF at much more dilute concentrations than could heretofore be used in most prior coagulation assays such as prior PT and thromboplastin assays. In particular, a plasma coagulation assay conducted in accord with the invention can initiate extrinsic coagulation with about 1 nM or less TF. The present methods also desirably prolong clotting times by inhibiting or eliminating unwanted contact coagulation. As discussed, contact coagulation often masks physiologically relevant coagulation reactions. Thus, the invention provides methods of assaying coagulation that more closely resemble in vivo coagulation. In particular, addition of CTI to the assay makes that specific assay less dependent on high levels of TF and more representative of in vivo clotting.
The invention provides further advantages. For example, the invention provides sensitivity to Factor XI at low TF concentrations, e.g,. below about 25 pM. Thus, the methods allow effective monitoring of coagulation factors for which analysis has been heretofore difficult to achieve.
Importantly, the invention provides an effective and reproducible means of assaying coagulation in patients receiving anti-coagulant therapy. For example, addition of CTI to a coagulation assay in accord with the invention provides more uniformity in INR value determinations. In particular, prior practice has made it difficult to obtain accurate INR values for those patients receiving anti-coagulants such as coumadin. The present invention allows for more uniformity by making linear relationships easier to obtain, ie., between clotting times and INR values.
The invention provides still further advantages. For example, particular methods described herein use citrated plasma to remove calcium. Addition of CTI to the recalcified plasma enhances use of that plasma by reducing or eliminating interference from contact activation. Thus, practice of the invention enhances the initiation of clotting by TF rather than calcium. In contrast, prior practice has generally dictated initiation of clotting by calcium addition. Thus, the present invention facilitates use of coagulation assays and especially the dilute TF assays by improving sample handling.
These and related advantages of the invention enhance practice of the present methods in a variety of settings such as those associated with commercial, medical, clinical, hospital or research applications.
As noted, the present invention is flexible and can be adapted to suit intended use. For example, the present methods can be modified if desired to combine platelet poor plasma with contact pathway inhibitors, thereby allowing advance recalcification of the plasma while avoiding opportunities for spurious contact-activated coagulation.
As also noted, the present invention can provide effective and reproducible inhibition of contact clotting in a blood product that is frozen or has been subjected to multiple freeze-thaw cycles. Thus, specific methods described herein can also be employed to facilitate more efficient handing and use of frozen or previously frozen blood products and particularly plasma such as platelet-deficient plasma.
Additional aspects and advantages of the invention are discussed infra.